Rosarugosides A and D from Rosa rugosa Flower Buds: Their Potential Anti-Skin-Aging Effects in TNF-α-Induced Human Dermal Fibroblasts

This present study investigated the anti-skin-aging properties of Rosa rugosa. Initially, phenolic compounds were isolated from a hot water extract of Rosa rugosa’s flower buds. Through repeated chromatography (column chromatography, MPLC, and prep HPLC), we identified nine phenolic compounds (1–9), including a previously undescribed depside, rosarugoside D (1). The chemical structure of 1 was elucidated via NMR, HR-MS, UV, and hydrolysis. Next, in order to identify bioactive compounds that are effective against TNF-α-induced NHDF cells, we measured intracellular ROS production in samples treated with each of the isolated compounds (1–9). All isolates reduced the level of ROS at a concentration of 10 μM. Particularly, two depsides—rosarugosides A and D (2 and 1)—significantly inhibited ROS expression in TNF-α-induced NHDFs compared to the other phenolic compounds. Subsequently, the production of MMP-1 and procollagen type Ι α1 by these two depsides was examined. Remarkably, rosarugoside A (2) significantly decreased MMP-1 secretion at all concentrations. In contrast, rosarugoside D (1) regulated the expression of procollagen type Ι α1. These findings collectively suggest that Rosa rugosa extracts and their isolated compounds, rosarugosides A (2) and D (1), hold significant potential for protecting against aging and skin damage. Overall, these findings suggest that Rosa rugosa extracts and their isolated compounds, rosarugosides A (2) and D (1), have the potential to prevent and protect against aging and skin damage, although more specific quantitative analysis is needed.


Introduction
Rosa rugosa (family Rosaceae), commonly known as rugosa rose, is native to East Asia and is now widely cultivated worldwide [1].It is well known for its aromatic flowers and is often used to manufacture rose hip oil, which contains sweet, fragrant, and bioactive volatile oils [2].It also is commonly used to make teas, wines, and jams, highlighting its many applications in food [3].In addition to its culinary applications, the flower buds of R. rugosa have long been used in traditional oriental medicine, particularly for their roles in the expansion of blood vessels and enhancement of microcirculation.These functions have prompted recent scientific interest in the pharmacological activities and bioactive compounds of R. rugosa [4].Previous phytochemical studies on R. rugosa have demonstrated that its flowers are highly abundant in phenolic compounds, such as flavonoids, tannins, and anthocyanins, which are recognized for their antioxidant properties [5].Among its unique phytochemicals, which is to say rugosin A-G, a series of hydrolyzable tannins characterized by valoneoyl groups have been isolated from the flower petals of R. rugosa.These Plants 2024, 13, 1266 2 of 13 have revealed their potential anti-inflammatory, antioxidant, anti-cancer, and antimicrobial properties [6][7][8][9][10].Additionally, we previously reported on unreported depsides with 2-O-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxyphenylacetic acid skeletons from R. rugosa, which were shown to improve schizophrenia-like symptoms, suggesting the pharmacological utility of R. rugosa [11].Yu et al. reported that an extract from Rosa rugosa had antioxidant and anti-inflammatory effects, displayed collagenase activity, and inhibited melanin formation in B16F10 mouse melanoma cells [12].Also, it was reported to have anti-aging, whitening, and moisturizing properties [13][14][15][16].In this study, we focused on R. rugosa extracts as they were expected to be effective in assisting the anti-aging process of the skin.
Aging, a natural biological process influenced by both intrinsic and extrinsic factors, encompasses a decline in cellular and tissue function, alterations in gene expression, and metabolic changes [17].These aging phenomena include the functional decline of cells and tissues, changes in gene expression, and changes in metabolic processes.Reactive oxygen species (ROS) play a crucial role in intracellular signaling and homeostasis at low levels, maintaining balance inside cells at low concentrations [18][19][20].However, excessive ROS production, caused by external factors, can lead to oxidative stress, which can accelerate aging and contribute to the occurrence of related diseases [21,22].In particular, oxidative stress also affects the health and beauty of the skin and is associated with wrinkles and the loss of elasticity [23].
Another important factor associated with wrinkles and the loss of elasticity in the skin is proteolytic enzymes, called matrix metalloproteinases (MMPs) [24].Among the various types of MMPs, MMP-1 promotes collagen breakdown, contributing to fine wrinkles and the loss of elasticity in the skin [25][26][27].
Collagen is found in a variety of connective tissues, including the skin, cartilage, and tendons, where it plays an important role, supporting the structure and maintaining elasticity [28,29].The increased breakdown of collagen due to aging and the effects of the external environment can reduce the capacity of the skin and lead to the formation of wrinkles [30].The interaction between ROS, MMP-1, and collagen forms a core mechanism of skin aging, and regulating this process is important for maintaining skin health.
Previous research has reported that the ethanol extract of R. rugosa flowers has antioxidant characteristics owing to its bioactive phenolic components, as demonstrated by the DPPH radical scavenging assay and the inhibitory activity of tyrosinase [31].In our preliminary investigation, hot water extracts of R. rugosa flower buds suppressed ROS production in tumor necrosis factor-alpha (TNF-α)-stimulated normal human dermal fibroblasts (NHDFs), suggesting potential anti-aging properties.Therefore, this study aimed to validate the anti-aging properties of hot water extracts of R. rugosa flowers and to identify the bioactive compounds present in the extract.

The Effects of Hot Water Extracts of R. rugosa Flower Buds and Compounds 1-9 on NHDF Cell Viability
The cytotoxicity of the samples was further investigated in order to determine the effects of the extract and isolated compounds 1-9 on NHDFs.The investigation showed that the extract and all the isolates had no toxicity to NHDFs at concentrations below 10 µM (Figure 3).Based on this, ROS screening was performed at concentrations of ≤10 µM.

The Effects of Hot Water Extracts of R. rugosa Flower Buds and Compounds 1-9 on NHDF Cell Viability
The cytotoxicity of the samples was further investigated in order to determine the effects of the extract and isolated compounds 1-9 on NHDFs.The investigation showed that the extract and all the isolates had no toxicity to NHDFs at concentrations below 10 µM (Figure 3).Based on this, ROS screening was performed at concentrations of ≤10 µM.

The Effects of Hot Water Extracts of R. rugosa Flower Buds and Compounds 1-9 on NHDF Cell Viability
The cytotoxicity of the samples was further investigated in order to determine the effects of the extract and isolated compounds 1-9 on NHDFs.The investigation showed that the extract and all the isolates had no toxicity to NHDFs at concentrations below 10 µM (Figure 3).Based on this, ROS screening was performed at concentrations of ≤10 µM.

The Effects of Hot Water Extracts of R. rugosa Flower Buds and Compounds 1-9 on NHDF Cell Viability
The cytotoxicity of the samples was further investigated in order to determine the effects of the extract and isolated compounds 1-9 on NHDFs.The investigation showed that the extract and all the isolates had no toxicity to NHDFs at concentrations below 10 µM (Figure 3).Based on this, ROS screening was performed at concentrations of ≤10 µM.UV activates TNF-α receptors on the surface of skin cells, and increased TNF-α levels induce intracellular ROS production [38].Therefore, we screened the effects of the R. rugosa extracts and compounds 1-9 on intracellular TNF-α-induced ROS production in NHDFs.In comparison to the control group, ROS generation in the group treated with TNF-α showed a significant increase (2.57 ± 0.02-fold; p < 0.001).Conversely, in the group treated with R. rugosa extracts, the level of TNF-α-induced ROS production was found to be reduced.Similarly, most of the nine compounds also suppressed the level of ROS at a concentration of 10 µM (Figure 4

The Effects of Rosarugosides D (1) and A (2) on TNF-α-Induced Intracellular ROS Production in NHDFs
To compare rosarugoside D (1), which significantly inhibited ROS generation in a previous screening, and rosarugoside A (2), which was similar in structure to compound

Discussion
The skin is mostly affected by external environmental factors, and exposure to ultraviolet rays, among other factors, can damage the skin [40].The skin is largely composed of the epidermis and dermis.The epidermis is the outermost layer of the skin and contains melanocytes, which primarily determine skin color [41].Additionally, the epidermis protects the body from the external environment and supplies nutrition to the skin through substance exchange [42,43].The dermis is located below the epidermis, and several functional elements, such as blood vessels, nerves, and sweat glands, are located in the dermis [44].In addition, proteins such as collagen and elastic fibers are abundantly distributed, maintaining the skin's elasticity and providing structural support [45].
Exposure to ultraviolet rays is necessary to induce the production of vitamin D, but long-term exposure to ultraviolet rays damages the dermis and fibroblasts and reduces the collagen content [46,47].Additionally, ultraviolet rays induce pro-inflammatory cytokines such as TNF-α [48].TNF-α causes inflammatory reactions in the skin and the excessive production of intracellular ROS [38,49].This ROS production induces the expression of MMPs.When collagen is broken down by MMPs, the ECM that maintains the elasticity of the skin and its structure collapses, causing sagging, wrinkles, and the loss of skin elasticity [50].In addition, ROS are associated with various skin diseases such as eczema, contact dermatitis, melanoma, and skin cancer; therefore, suppressing TNF-α-induced ROS secretion is an important factor in preventing skin aging and disease [21, [51][52][53].2) for 1 h, and treated with TNF-α at 20 ng/mL for 24 h.The effects of the compounds were investigated using an ELISA kit.The data are depicted as mean ± S.E.M (n = 3).### p < 0.001 control group versus TNF-α-induced group.*p < 0.05, and ***p < 0.001 sample treatment group versus TNF-α-induced group.

Discussion
The skin is mostly affected by external environmental factors, and exposure to ultraviolet rays, among other factors, can damage the skin [40].The skin is largely composed of the epidermis and dermis.The epidermis is the outermost layer of the skin and contains melanocytes, which primarily determine skin color [41].Additionally, the epidermis protects the body from the external environment and supplies nutrition to the skin through substance exchange [42,43].The dermis is located below the epidermis, and several functional elements, such as blood vessels, nerves, and sweat glands, are located in the dermis [44].In addition, proteins such as collagen and elastic fibers are abundantly distributed, maintaining the skin's elasticity and providing structural support [45].
Exposure to ultraviolet rays is necessary to induce the production of vitamin D, but long-term exposure to ultraviolet rays damages the dermis and fibroblasts and reduces the collagen content [46,47].Additionally, ultraviolet rays induce pro-inflammatory cytokines such as TNF-α [48].TNF-α causes inflammatory reactions in the skin and the excessive production of intracellular ROS [38,49].This ROS production induces the expression of MMPs.When collagen is broken down by MMPs, the ECM that maintains the elasticity of the skin and its structure collapses, causing sagging, wrinkles, and the loss of skin elasticity [50].In addition, ROS are associated with various skin diseases such as eczema, contact dermatitis, melanoma, and skin cancer; therefore, suppressing TNF-α-induced ROS secretion is an important factor in preventing skin aging and disease [21, [51][52][53].
Today, consumers are becoming more interested in health and skin aging, and the antiaging cosmetics industry is attracting attention.Natural products are attracting attention in research on traditional and alternative medicine [54].Ascorbic acid, polyphenols, and other natural products are antioxidants that prevent intrinsic and extrinsic aging [55].Additionally, the awareness of the health benefits of compounds obtained from natural sources is increasing, exemplified by the development of cosmetics using natural ingredients such as green tea extract, quercetin, and resveratrol [56].
The antioxidant efficacy of R. rugosa, one of the natural materials, was also determined by analyzing ROS production from extracts and compounds.As a result, nine compounds taken from the hot extract of R. rugosa flowers were found to be effective in suppressing the increase in ROS production induced by TNF-α (Figure 4).Among these, rosarugoside D (1), a newly isolated and unreported depside, and rosarugoside A (2), which has a similar structure to rosarugoside D (1) except for the presence of an O-glucose at position C-8, were selected, and the production of MMP-1 and procollagen type I α1 was investigated.
UV and TNF-α induce various types of MMPs in the skin, such as MMP-1 (collagenase), MMP-3 (gelatinase), and MMP-9 (stromelysin-1) [57][58][59].Among these, MMP-1 is a procollagen-degrading enzyme [60].Procollagen type I α1 is the precursor molecule of type I collagen [61].Collagen type I makes up 90% of the human body and is mainly found in skin, bones, and tendons [62].Additionally, the induction of type I collagen synthesis in human fibroblasts is an important criterion for evaluating the function of cosmetic ingredients [63].We investigated the increased levels of MMP-1 and decreased levels of procollagen type I α1 in rosarugosides D (1) and A (2) in TNF-α-induced NHDFs.We found that rosarugoside D (1) showed weak efficacy in terms of inhibiting MMP-1, but tended to restore procollagen type I α1.On the other hand, rosarugoside A (2) had a strong inhibitory effect on MMP-1 at all concentrations, but did not restore procollagen type I α1 (Figure 6).
These results demonstrate the potential that the pharmacological activities of R. rugosa flower buds and their depsides, rosarugosides D (1) and A (2), have in terms of preventing and protecting against TNF-α-induced skin damage.However, this study has some limitations.For example, ROS overexpression due to TNF-α activates the c-Jun/activator protein-1 (AP-1), nuclear factor kappa B (NF-κB), and transforming growth factor-beta (TGF-β) signaling pathways [64].Through these pathways, it secretes various MMPs and induces inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines [25,65].However, this mechanism was not investigated in detail in this study.Additional studies are needed to understand the protective effects of rosarugosides D (1) and A (2).

Plant Material
Dried flower buds of R. rugosa were purchased from the Kyungdong Market, Seoul, Republic of Korea, in August 2016.The plant material was authenticated by Prof. Dae Sik Jang, and a reference sample (RORU2-2016) was deposited at the College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea.

Extraction and Isolation
We extracted products from the dried flowers of R. rugosa (1.1 kg) by subjecting them to hot water (10 L) for 2 h twice.The crude extract (150.0 g, RORU-Ext) was homogeneously mixed with Diaion HP-20 and water.The solution was then evaporated in vacuo at 45 • C. Subsequently, the powered mixture was fractionated using open-column chromatography (CC) after packing the apparatus with the same resin, generating nine fractions (R1~R9).Subsequently, a Sephadex LH-20 CC (acetone-water = 2:8~6:4) was employed to separate 143.9

Identification of Sugar
To conduct absolute structural elucidation of sugars, acid hydrolysis was performed on compound 1 (1.0 mg) by dissolving it in 2 M trifluoroacetic acid (TFA) and heating the solution at 60 • C for 2 h.After hydrolysis, the reaction mixtures were concentrated and then subjected to liquid-liquid extraction using water and butanol.The aqueous layer was further processed by dissolving it in a solution of 10 mg/mL L-cysteine methyl ester and pyridine, and the mixture was incubated at 60 • C for 1 h in a water bath.Then, 20.0 µL of o-tolyl isothiocyanate was added, and the reaction was allowed to proceed for an hour at the same temperature in order to derivatize the sugars.
Chromatographic separation of the derivatized sugars was achieved using a Vanquish UHPLC-DAD.This was equipped with Hypersil GOLD C18 column (150 × 2.1 mm, 1.9 µm, Thermo Scientific, Waltham, MA, USA) and maintained at 35 • C. The analysis was conducted over 20 min under isocratic conditions, employing a mobile phase composed of 0.5% formic acid in water and 0.5% formic acid in acetonitrile, mixed at a ratio of 80:20.The identification of D-glucose was confirmed by comparing the retention times (11.4 min) and mass spectral data obtained using an LTQ-XL ion trap mass spectrometer (Thermo Scientific) with those of authentic standards, ensuring the accurate characterization of the sugars present in the samples.

Cell Culture
Normal human dermal fibroblasts (NHDFs) were grown on a medium at 37 • C in a humidified atmosphere of 5% CO 2 .Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, was used as the culture medium.

Cell Viability
Cell viability was assessed in 96-well culture plates (1 × 10 4 cells/well) using the EZ-Cytox solution.To check the toxicity of the sample, each compound was diluted in a serum-free medium to a concentration of 1-100 µM and then treated.After incubation for 24 h, the supernatant was removed and 10% EZ-Cytox in a serum-free medium was added to the samples.The mixture was then incubated at 37 • C for 1 h.Subsequently, optical density was measured using a microplate reader.

Intracellular Reactive Oxygen Species (ROS)
Intracellular ROS production was measured in 96-well black culture plates (1 × 10 4 cells/well) using a 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (DCFDA) assay.NHDFs were exposed to the extract or compounds for 1 h and then stimulated with 20 ng/mL TNF-α and 10 µM DCFDA for 15 min at 37 • C. Subsequently, the treated NHDFs were washed with PBS after removing the supernatant.Finally, ROS production was measured using a microplate reader, assessing excitation (485 nm) and emission (530 nm) wavelengths.

Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA was performed on 48-well culture plates (2 × 10 4 cells/well) using MMP-1 and the procollagen type I α1 ELISA Kit.NHDFs were exposed to compounds for 1 h and stimulated with 20 ng/mL TNF-α for 24 h at 37 • C.After that, the experiment was performed using the supernatant according to the manufacturer's instructions.Optical density was measured using a microplate reader at 450 nm.

Statistical Analysis
The data are presented as the mean ± standard deviations (SD) and as the standard error of the mean (S.E.M).Differences between treatment groups were analyzed using one-way analysis of variance, and significance was defined by Tukey's test as a level of p < 0.05.The data were analyzed using GraphPad Prism version 8.0.1 (GraphPad Software Inc., La Jolla, CA, USA).

14 Figure 3 .
Figure 3.The effects of hot water extracts of R. rugosa flower buds (Ext.) and compounds 1-9 on NHDF cell viability.The cells were then treated with (1-10 µM) concentrations of the compound for 24 h.The effects of the compounds on cell viability were evaluated using an EZ-Cytox solution.The data are presented as the mean ± SD (n = 3).

Figure 3 .
Figure 3.The effects of hot water extracts of R. rugosa flower buds (Ext.) and compounds 1-9 on NHDF cell viability.The cells were then treated with (1-10 µM) concentrations of the compound for 24 h.The effects of the compounds on cell viability were evaluated using an EZ-Cytox solution.The data are presented as the mean ± SD (n = 3).
The Effect of Hot Water Extracts of R. rugosa Flower Buds and Compounds 1-9 on TNF-α-Induced Intracellular ROS Production in NHDFs